During times of fasting, energy stored in the adipocyte as triglyceride is released for use by muscle, liver and other tissues. This process, termed lipolysis, is highly regulated by hormonal, neural and nutritional inputs. There is subtantial evdence that resistance to the actions of insulin to antagonize lipolysis is an early and important step in the development of type 2 diabetes mellitus and the metabolic syndrome. The intracellular second messenger most frequently used to convey the signal for increased lipolysis is cyclic AMP, which accumulates in reponse to beta-adrenergic agents and activates protein kinase A. Much is known about signaling downstream of protein kinase A, including a major substrate, perilipin, present on the triglyceride lipid droplet. Insulin antagonizes the process of lipolysis, and there is a generally accepted model to explain the acute suppression of adipocyte lipolysis, though it has not been studied in detail. The canonical model of insulin signaling describes phosphoinositide 3'-kinase-dependent activation of Akt (protein kinase B), which then phosphorylates and stimulates phosphodiesterase 3B (PDE3B), leading to a decrease in intracellular cyclic AMP. Previous work from the laboratory of the principal investigator has revealed, however, an alternative pathway of insulin action that comes into play upon sub-maximal stimulation of lipolysis. Preliminary data indicate that this signaling cascade functions independently of Akt and that the critical signaling events occur on the surface the lipid droplet. The studies described in this grant proposal will formally test the hypothesis of an alternative pathway for anti-lipolytic signaling. Experiments include the use of FRET biosensors to determine the precise intracellular sites of PKA activation in cultured adipocytes and the identification of scaffolding proteins that target regulatory molecules ot the lipid droplet. The requirement for Akt and PDE3B phosphorylation in the suppression of lipolysis will be tested for the first time by genetic loss of function experiments. Lastly, the contribution of this novel pathway for the suppression of fat cell lipolysis will be assessed for physiological relevance through in vivo experiments in genetically modified mice. PUBLIC HEALTH RELEVANCE: Type 2 diabetes mellitus and the metabolic syndrome have reached epidemic proportions in the United States. A critical pathophysiological event in the development of these diseases is the inability of insulin to suppress fatty acid release from adipose tissue, thus leading to abnormal deposition of fat in other tissues. This proposal seeks to understand in detail the pathway by which insulin prevents fatty acid release from fat cells.